E.Z.N.A. Plasmid DNA Mini Kit I

Transcription

1 E.Z.N.A. Plasmid DNA Mini Kit I D preps Q - Spin D preps Q - Spin D preps Q - Spin D preps V - Spin D preps V - Spin D preps V - Spin E.Z.N.A. Plasmid DNA Mini Kit II D preps V - Spin D preps V - Spin D preps V - Spin May 2013

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3 E.Z.N.A. Plasmid DNA Mini Kit I E.Z.N.A. Plasmid DNA Mini Kit II Table of Contents Introduction...2 Yield and Quality of DNA...3 Illustrated Protocol...4 Kit Contents...5 Preparing Reagents/Storage and Stability...6 Guidelines for Vacuum Manifold...7 Recommended Settings...8 Plasmid DNA Mini Kit I Centrifugation Protocol...10 Plasmid DNA Mini Kit I Vacuum Protocol...13 Plasmid DNA Mini Kit II Centrifugation Protocol...17 Low Copy Number Plasmid Protocol...21 Troubleshooting Guide...22 Ordering...24 Manual Revision: May

4 Introduction The E.Z.N.A. family of products is an innovative system that radically simplifies the extraction and purification of nucleic acids from a variety of sources. The key to this system is the HiBind matrix that specifically, but reversibly, binds DNA or RNA under optimized conditions allowing proteins and other contaminants to be removed. Nucleic acids are easily eluted with deionized water or low salt buffer. The E.Z.N.A. Plasmid DNA Mini Kits combine the power of HiBind technology with the time-tested consistency of alkaline-sds lysis of bacterial cells to deliver high-quality DNA in less than 30 minutes. HiBind DNA Mini Columns facilitate the binding, washing, and elution steps thus enabling multiple samples to be processed simultaneously. Typically, a 1.5 ml overnight culture in LB medium produces 3-12 μg plasmid DNA; although yields may vary according to plasmid copy number, E. coli strain, and growth conditions. The E.Z.N.A. Plasmid DNA Mini Kit I is used to isolate plasmid DNA from 1-5 ml cultures. The E.Z.N.A. Plasmid DNA Mini Kit II can isolate μg plasmid DNA from ml cultures when using high copy plasmids. Purified plasmid DNA can be directly used for most downstream applications including automated fluorescent DNA sequencing and restriction enzyme digestion. New In this Edition HB Buffer has been replaced by HBC Buffer. Isopropanol is required and supplied by the user. Equilibration Buffer is no longer included with this kit. An optional Column Equilibration Protocol has been added to the protocol for your convenience. Equilibration Buffer is replaced with 3M NaOH provided by the user. Q-spin column vs. V-spin column The E.Z.N.A. Plasmid DNA Mini Kit is available with two different types of columns. V-spin columns have an attached cap, while Q-spin columns are capless. The columns are otherwise identical in use and application. Either column can be used with either the vacuum or centrifugation protocol. Protocols The E.Z.N.A. Plasmid DNA Mini Kits are designed for fast and efficient processing. Depending on the protocol, the E.Z.N.A. Plasmid Mini Kits can be used with any microcentrifuge or vacuum manifold with standard luer connectors. 2

5 Yield and Quality of DNA Determine the absorbance of an appropriate dilution (20- to 50-fold) of the sample at 260 nm and then at 280 nm. The DNA concentration is calculated as follows: DNA concentration = Absorbance (Dilution Factor) µg/ml A ratio greater than 1.8 indicates greater than 90% nucleic acid. Alternatively, quantity (as well as quality) sometimes can be determined best by agarose gel/ethidium bromide electrophoresis by comparison to DNA samples of known concentrations. Typically, the majority of the DNA eluted is in monomeric supercoil form, though concatemers may also be present. Plasmid Copy Number and Expected Yield Yield and quality of the plasmid DNA obtained depends on a number of factors including plasmid copy number, size of insert, host strain, culture volume, culture medium, and binding capacity of the kit. Of these factors, the vector copy number, culture volume, and kit binding capacity are most important. Plasmid copy number ranges from one copy to several hundred copies per cell as dictated by their origin of replication. Very large plasmids often display a very low copy number per cell. The expected yield of 5 ml overnight cultures (LB medium) with the E.Z.N.A. Plasmid Mini Kit are indicated in the following table. Sample yields from a 5 ml starting culture. Plasmid Replicon Copy Number Expected Yield puc vectors pmbi µg pbluescript vectors ColE µg pgem vectors pmb µg pbr322 and its derivatives pmb µg ColE14 ColE µg PACYC and its derivatives p15a µg psc101 and its derivatives psc101 ~5 0.5 µg pgem pmb µg 3

6 Spin Protocol Vacuum/Spin Protocol Pellet by Centrifugation Pellet by Centrifugation Resuspend and Lyse Resuspend and Lyse Neutralize Neutralize Clear Lysate Clear Lysate Transfer Lysate to HiBind DNA Mini Column Transfer Lysate to HiBind DNA Mini Column Bind Bind Wash 3X Wash 3X Dry Dry Elute Elute 4

7 Kit Contents Plasmid Mini Kit I D D D D D D Preps HiBind DNA Mini Columns ml Collection Tubes Solution I 3 ml 20 ml 60 ml Solution II 3 ml 20 ml 60 ml Solution III 3 ml 20 ml 80 ml HBC Buffer 4 ml 25 ml 80 ml DNA Wash Buffer 1.5 ml 15 ml 3 x 25 ml RNase A Pre-Added 100 µl 400 µl Elution Buffer 1.5 ml 10 ml 30 ml User Manual P P P Plasmid Mini Kit II D D D Preps HiBind DNA Mini Columns II ml Collection Tubes Solution I 5 ml 30 ml 120 ml Solution II 5 ml 30 ml 120 ml Solution III 5 ml 40 ml 2 x 80 ml HBC Buffer 4 ml 25 ml 80 ml DNA Wash Buffer 1.5 ml 15 ml 3 x 25 ml RNase A Pre-Added 100 µl 400 µl Elution Buffer 1.5 ml 10 ml 30 ml User Manual P P P 5

8 Preparing Reagents 1. Add the vial of RNase A to the bottle of Solution I and store at 2-8 C. (50 and 200 prep size only). 2. Dilute DNA Wash Buffer with 100% ethanol as follows and store at room temperature. Kit D D D D D D D D D % Ethanol to be Added 6 ml 60 ml 100 ml 3. Dilute HBC Buffer with isopropanol as follows and store at room temperature. Kit D D D D D D D D D Isopropanol to be Added 1.6 ml 10 ml 32 ml 4. Check Solution II and Solution III for precipitation before use. Redissolve any precipitation by warming to 37 C. 6 Storage and Stability All of the E.Z.N.A. Plasmid DNA Mini Kit I and Plasmid DNA Mini Kit II components are guaranteed for at least 12 months from the date of purchase when stored as follows. Solution I (once RNase A is added) should be stored at 2-8 C. All other materials should be stored at room temperature. Solution II must be tightly capped when not in use.

9 Guidelines for Vacuum Manifold The following is required for use with the Vacuum/Spin Protocol: A) Vacuum Manifold (We recommend Omega Bio-tek s VAC-08) Other Compatible Vacuum Manifolds: Qiagen QIAvac24, Sigma AldrichVM20, Promega Vacman, or manifold with standard Luer connector B) Vacuum Flask C) Vacuum Tubing D) Vacuum Source (review tables below for pressure settings) Manifold Recommended Pressure (mbar) VAC to -600 Conversion from millibars: Multiply by: Millimeters of mercury (mmhg) 0.75 Kilopascals (kpa) 0.1 Inches of mercury (inch Hg) Torrs (Torr) 0.75 Atmospheres (atmos) Pounds per Square Inch (psi) Vacuum Setup: Omega Bio-tek s VAC-08 C) Vacuum Tubing A) Vacuum Manifold D) Vacuum Source B) Vacuum Flask 7

10 Recommended Settings Growth and Culture of Bacteria Bacterial Strain Selection It is strongly recommended that an end A negative strain of E. coli be used for routine plasmid isolation. Examples of such strains include DH5α, DH1, and C600. These host strains yield high-quality DNA with E.Z.N.A. Plasmid DNA Mini Kit Protocols. XL1-Blue, although a slower growing strain is also recommended due to its yield of high-quality DNA. Host strains derivatives from HB101 such as TG1 and the JM100 series release large amounts of carbohydrates during lysis, which may inhibit enzyme activity when not completely removed. Some strains may also lower DNA quality due to having high levels of endonuclease activity, and therefore are not recommended (i.e. JM101, JM110, HB101). One may reduce the amount of culture volume or double the volumes of Solution I, Solution II, and Solution III, if problems are encountered with strains such as TG1 and Top10F. Inoculation Bacterial cultures for plasmid preparations should always be grown from a single colony picked from a freshly streaked plate. Subculturing directly from glycerol stock or liquid cultures may lead to uneven yields or plasmid loss. Optimal results are obtained by using one single isolated colony from a freshly transformed or freshly streaked plate to inoculate an appropriate volume of starter culture containing the appropriate antibiotic, and then incubated for at 37 C with vigorous shaking (~300 rpm; shaking incubator). Note: Aeration is very important. The culture volume should not exceed 1/4 the volume of the container. Culture Media The E.Z.N.A. Plasmid DNA Mini Kits are specially designed for use with cultures grown in Luria Bertani (LB) medium. Richer broths such as TB(Terrific Broth) or 2xYT lead to high cell densities that can overload the purification system, and therefore are not recommended. If rich media has to be used, growth times have to be optimized, and the recommended culture volumes must be reduced to match the capacity of the HiBind DNA Mini Column. Note: As culture ages, DNA yield may begin to decrease due to cell death and lysis within the culture. 8

11 Recommended Settings Culture Volume and Cell Density Do Not Exceed Maximum Recommended Culture Volumes For optimal plasmid yields, the starting culture volume should be based on culture cell density. A bacterial density between 2.0 and 3.0 at OD 600 is recommended. When using nutrient-rich media, care should be taken to ensure that the cell density does not exceed an OD 600 of 3.0. Using a high-density culture outside of the recommended OD range may overload the purification system. 9

12 E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol E.Z.N.A. Plasmid DNA Mini Kit I Protocol - Spin Protocol All centrifugation should be performed at room temperature unless otherwise noted. For low copy number plasmids refer to Page 21. This protocol is designed to isolate plasmid DNA from E. coli grown in an overnight 1-5 ml LB culture. Materials and Equipment to be Supplied by User: 100% ethanol Isopropanol Microcentrifuge capable of at least 13,000 x g Nuclease-free 1.5 ml or 2 ml microcentrifuge tubes Culture tubes Optional: sterile deionized water Optional: water bath or incubator capable of 70 C Optional: 3M NaOH solution Before Starting: Heat Elution Buffer to 70 C if plasmid DNA is >10 kb Prepare DNA Wash Buffer, HBC Buffer, and Solution I according to the instructions in the Preparing Reagents section on Page 6 1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1-5 ml LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hours at 37 C with vigorous shaking (~ 300 rpm). Use a ml culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that an enda negative strain of E. coli be used for routine plasmid isolation. Examples of such strains include DH5a and JM Centrifuge at 10,000 x g for 1 minute at room temperature. 3. Decant or aspirate and discard the culture media. 4. Add 250 µl Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields. Note: RNase A must be added to Solution I before use. Please see the instructions in the Preparing Reagents section on Page 6. 10

13 E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol 5. Transfer suspension into a new 1.5 ml microcentrifuge tube. 6. Add 250 µl Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary. Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 minutes. Store Solution II tightly capped when not in use to avoid acidification from CO 2 in the air. 7. Add 350 µl Solution III. Immediately invert several times until a flocculent white precipitate forms. Note: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation. 8. Centrifuge at maximum speed ( 13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step. 9. Insert a HiBind DNA Mini Column into a 2 ml Collection Tube. Optional Protocol for Column Equilibration: 1. Add 100 µl 3M NaOH to the HiBind DNA Mini Column. 2. Centrifuge at maximum speed for seconds. 3. Discard the filtrate and reuse the collection tube. 10. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column. 11. Centrifuge at maximum speed for 1 minute. 12. Discard the filtrate and reuse the collection tube. 13. Add 500 µl HBC Buffer. Note: HBC Buffer must be diluted with isopropanol before use. Please see Page 6 for instructions. 11

14 E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol 14. Centrifuge at maximum speed for 1 minute. 15. Discard the filtrate and reuse collection tube. 16. Add 700 µl DNA Wash Buffer. Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. Please see Page 6 for instructions. 17. Centrifuge at maximum speed for 1 minute. 18. Discard the filtrate and reuse the collection tube. Optional: Repeat Steps for a second DNA Wash Buffer wash step. 19. Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix. Note: It is important to dry the HiBind DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications. 20. Transfer the HiBind DNA Mini Column to a clean 1.5 ml microcentrifuge tube. 21. Add μl Elution Buffer or sterile deionized water directly to the center of the column membrane. Note: The efficiency of eluting DNA from the HiBind DNA Mini Column is dependent on ph. If using sterile deionized water, make sure that the ph is around Let sit at room temperature for 1 minute. 23. Centrifuge at maximum speed for 1 minute. Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration Store DNA at -20 C.

15 E.Z.N.A. Plasmid DNA Mini Kit I Vacuum Protocol E.Z.N.A. Plasmid Mini Kit I Protocol - Vacuum Protocol All centrifugation should be performed at room temperature unless otherwise noted. For low copy number plasmids refer to Page 21. This protocol is designed to isolate plasmid DNA from E. coli grown in an overnight 1-5 ml LB culture. See Page 7 for guidelines on preparing the vacuum manifold used in this protocol. Materials and Equipment to be Supplied by User: Vacuum Manifold 100% ethanol Isopropanol Microcentrifuge capable of at least 13,000 x g Nuclease-free 1.5 ml or 2 ml microcentrifuge tubes Appropriate centrifuge and centrifuge tube for Step 1 Optional: sterile deionized water Optional: water bath or incubator capable of 70 C Optional: 3M NaOH solution Before Starting: Heat Elution Buffer to 70 C if plasmid DNA is >10 kb Prepare DNA Wash Buffer, HBC Buffer, and Solution I according to the instructions in the Preparing Reagents section on Page 6 1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1-5 ml LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hr at 37 C with vigorous shaking (~ 300 rpm). Use a ml culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that an enda negative strain of E. coli be used for routine plasmid isolation. Examples of such strains include DH5a and JM Centrifuge at 10,000 x g for 1 minute at room temperature. 3. Decant or aspirate and discard the culture media. 13

16 E.Z.N.A. Plasmid DNA Mini Kit I Vacuum Protocol 4. Add 250 µl Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields. Note: RNase A must be added to Solution I before use. Please see the instructions in the Preparing Reagents section on Page Transfer suspension into a new 1.5 ml microcentrifuge tube. 6. Add 250 µl Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary. Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 minutes. Store Solution II tightly capped when not in use to avoid acidification from CO 2 in the air. 7. Add 350 µl Solution III. Immediately invert several times until a flocculent white precipitate forms. Note: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation. 8. Centrifuge at maximum speed ( 13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step. 9. Prepare the vacuum manifold according to manufacturer s instructions. 10. Connect the HiBind DNA Mini Column to the vacuum manifold. Optional Protocol for Column Equilibration: 1. Add 100 µl 3M NaOH to the HiBind DNA Mini Column. 2. Turn on the vacuum source to draw the NaOH through the column. 3. Turn off the vacuum. 11. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column. 14

17 E.Z.N.A. Plasmid DNA Mini Kit I Vacuum Protocol 12. Turn on the vacuum source to draw the sample through the column. 13. Turn off the vacuum. 14. Add 500 µl HBC Buffer. Note: HBC Buffer must be diluted with isopropanol before use. Please see Page 6 for instructions. 15. Turn on the vacuum source to draw the buffer through the column. 16. Turn off the vacuum. 17. Add 700 µl DNA Wash Buffer. Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. Please see Page 6 for instructions. 18. Turn on the vacuum source to draw the buffer through the column. 19. Turn off the vacuum. 20. Repeat Steps for a second DNA Wash Buffer wash step. 21. Transfer the HiBind DNA Mini Column to a 2 ml Collection Tube. 22. Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix. Note: It is important to dry the HiBind DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications. 15

18 E.Z.N.A. Plasmid DNA Mini Kit I Vacuum Protocol 23. Transfer the HiBind DNA Mini Column to a clean 1.5 ml microcentrifuge tube. 24. Add μl Elution Buffer or sterile deionized water directly to the center of the column membrane. Note: The efficiency of eluting DNA from the HiBind DNA Mini Column is dependent on ph. If using sterile deionized water, make sure that the ph is around Let sit at room temperature for 1 minute. 26. Centrifuge at maximum speed for 1 minute. Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration. 27. Store DNA at -20 C. 16

19 E.Z.N.A. Plasmid DNA Mini Kit II Spin Protocol E.Z.N.A. Plasmid Mini Kit II Protocol - Spin Method All centrifugation should be performed at room temperature unless otherwise noted. For low copy number plasmids refer to Page 21. This protocol is designed to isolate plasmid DNA from E. coli grown in ml LB culture. Materials and Equipment to be Supplied by User: 100% ethanol Isopropanol Microcentrifuge capable of at least 13,000 x g Nuclease-free 2 ml microcentrifuge tubes Centrifuge capable of at least 5,000 x g with swing buckets Appropriate centrifuge tube for Step 1 Optional: sterile deionized water Optional: water bath or incubator capable of 70 C Optional: 3M NaOH solution Before Starting: Heat Elution Buffer to 70 C if plasmid DNA is >10kb Prepare DNA Wash Buffer, HBC Buffer, and Solution I according to the instructions in the Preparing Reagents section on Page 6 1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of ml(50 µg/ml) LB medium containing the appropriate selective antibiotic. Incubate for ~ hours at 37 C with vigorous shaking (~ 300 rpm). Use culture tube or a flask with a volume of at least 4 times the volume of the culture. It is strongly recommended that an end A negative strain of E. coli be used for routine plasmid isolation. Examples of such strains include DH5a and JM Centrifugation at 5,000 x g for 10 minutes at room temperature. 3. Decant or aspirate the medium and discard. 17

20 E.Z.N.A. Plasmid DNA Mini Kit II Spin Protocol 4. Add 500 µl Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields. Note: RNase A must be added to Solution I before use. Please see the instructions in the Preparing Reagents section on Page Transfer suspension into a new 2 ml microcentrifuge tube. 6. Add 500 µl Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary. Note: Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. Do not allow the lysis reaction to proceed more than 5 minutes. Store Solution II tightly capped when not in use to avoid acidification from CO 2 in the air. 7. Add 700 µl Solution III. Immediately invert several times until a flocculent white precipitate forms. Note: It is vital that the solution is mixed thoroughly and immediately after the addition of Solution III to avoid localized precipitation. 8. Centrifuge at maximum speed ( 13,000 x g) for 10 minutes at room temperature. A compact white pellet will form. Promptly proceed to the next step. 9. Insert a HiBind DNA Mini Column into a 2 ml Collection Tube. Optional Protocol for Column Equilibration: 1. Add 100 µl 3M NaOH to the HiBind DNA Mini Column. 2. Centrifuge at maximum speed for seconds. 3. Discard the filtrate and reuse the collection tube. 10. Transfer 700 µl cleared lysate from Step 8 by CAREFULLY aspirating it into the HiBind DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column. 11. Centrifuge at maximum speed for 1 minute. 18

21 E.Z.N.A. Plasmid DNA Mini Kit II Spin Protocol 12. Discard the filtrate and reuse the collection tube. 13. Repeat Steps until all cleared lysate has been transferred to the HiBind DNA Mini Column. 14. Add 500 µl HBC Buffer. Note: HBC Buffer must be diluted with isopropanol before use. Please see Page 6 for instructions. 15. Centrifuge at maximum speed for 1 minute. 16. Discard the filtrate and reuse collection tube. 17. Add 700 µl DNA Wash Buffer. Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. Please see Page 6 for instructions. 18. Centrifuge at maximum speed for 1 minute. 19. Discard the filtrate and reuse the collection tube. Optional: Repeat Steps for a second DNA Wash Buffer wash step. 20. Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix. Note: It is important to dry the HiBind DNA Mini Column matrix before elution. Residual ethanol may interfere with downstream applications. 19

22 E.Z.N.A. Plasmid DNA Mini Kit II Spin Protocol 21. Transfer the HiBind DNA Mini Column to a clean 1.5 ml microcentrifuge tube. 22. Add μl Elution Buffer or sterile deionized water directly to the center of the column membrane. Note: The efficiency of eluting DNA from the HiBind DNA Mini Column is dependent on ph. If using sterile deionized water, make sure that the ph is around Let sit at room temperature for 1 minute. 24. Centrifuge at maximum speed for 1 minute. Note: This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration. 25. Store DNA at -20 C. 20

23 Low Copy-Number Plasmid and BAC DNA Protocol E.Z.N.A. Plasmid Mini Kit Protocol - Low Copy Number Plasmid and BAC DNA Protocol Low copy number plasmids generally give µg DNA per ml overnight culture. For the isolation of plasmid DNA from low copy number plasmids (0.1-1 µg/ml culture) or low copy number plasmid (1-2 µg/ml culture) bacteria, use the following modified protocol. Note: The E.Z.N.A. Plasmid DNA Mini Kit l and the E.Z.N.A. Plasmid DNA Mini Kit ll come with enough Solution I, Solution II, and Solution III to perform the standard protocols. Additional Solution I, Solution II, and Solution III are needed to perform the Low Copy Number Plasmid and BAC DNA Protocol. These buffers can be purchased separately. See Page 24 for ordering information. 1. Increase the volume of starting culture from that of high copy number plasmids. Use 5-10 ml bacterial culture for the E.Z.N.A. Plasmid DNA Mini Kit l or ml bacterial culture for E.Z.N.A. Plasmid DNA Mini Kit ll. 2. Pellet the bacterial cells by centrifugation. 3. Decant or aspirate and discard the culture media. 4. Perform Steps 4-8 in the standard protocols with double the volumes of Solution I, Solution II, and Solution III. 5. Continue with Step 9 of the standard protocols by following the wash, drying, and elution steps. There is no need to increase the volumes of HBC Buffer, DNA Wash Buffer, or Elution Buffer. 21

24 Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise. For further assistance, please contact the technical support staff, toll free, at Possible Problems and Suggestions Low DNA yields Only use LB or YT medium containing ampicillin. Do not use more than 5 ml (high copy number plasmids) or 10 ml (low copy number plasmids) culture with the basic protocols. Poor cell lysis Cells may not have been dispersed adequately prior to the addition of Solution II. Vortex to completely resuspend the cells. Increase Solution II incubation time to obtain a clear lysate. Solution II, if not tightly closed, may need to be replaced. Culture is overgrown or not fresh Do not incubate cultures for more than 16 hours at 37ºC. Storage of cultures for extended periods prior to plasmid isolation is detrimental. Low elution efficiency The ph of Elution Buffer or water must be ph Low copy-number plasmid used Column matrix lost binding capacity during storage DNA Wash Buffer not diluted with ethanol HBC Buffer not diluted with isopropanol Such plasmids may yield as little as 0.1 μg DNA from a 1 ml overnight culture. Double the culture volume and follow the low copy number plasmid protocol on Page 21. Follow the Optional Protocol for Column Equilibration prior to transferring the cleared lysate to the HiBind DNA Mini Column. Add 100 µl 3M NaOH to the column prior to loading the sample. Centrifuge at 10,000 x g for 30 seconds. Discard the filtrate. No DNA eluted Prepare DNA Wash Buffer according to instructions on Page 6. Prepare HBC Buffer according to instructions on Page 6. 22

25 Troubleshooting Guide High molecular weight DNA contamination of product Over mixing of cell lysate upon addition of Solution II Culture overgrown Do not vortex or mix aggressively after adding Solution II. Overgrown cultures contain lysed cells and degraded DNA. Do not grow cell longer than 16 hours. Plasmid DNA floats out of well while loading agarose gel Ethanol was not completely removed from column following wash steps Centrifuge column as instructed to dry the column before elution. Absorbance of purified DNA does not accurately reflect quantity of the plasmid (A 260 /A 280 ratio is high or low) DNA Wash Buffer is diluted with ethanol containing impurities Plasmid DNA is contaminated with RNA; RNase A treatment is insufficient Background reading is high due to silica fine particulates Purification is incomplete due to column overloading Check the absorbance of the ethanol between 250 nm and 300 nm. Do not use ethanol with high absorbance. Traces of impurities may remain on the binding column after washing and contribute to the absorbance in the final product. Confirm that the RNase A Solution was added to Solution I prior to first use. The RNase A solution may degrade due to high temperatures (>65 C) or prolonged storage (> 6 months at room temperature). Spin the DNA sample at maximum speed for 1 minute; use the supernatant to repeat the absorbance readings. Reduce the initial volume of culture. 23

26 Ordering Information The following components are available for purchase separately. (Call Toll Free at ) Product DNase/RNase-free microcentrifuge tubes, 1.5 ml, 500/pk, 10 pk/cs DNase/RNase-free microcentrifuge tubes, 2.0 ml, 500/pk, 10 pk/cs Vacuum Manifold HiBind DNA Mini Columns (200) Solution I (250 ml) Solution II (250 ml) Solution III (250 ml) Elution Buffer (100 ml) DNA Wash Buffer (100 ml) RNase A (400 µl) Part Number SSI SSI VAC-08 DNACOL-02 PS001 PS002 PS003 PDR048 PS010 AC HiBind, E.Z.N.A., and MicroElute are registered trademarks of Omega Bio-tek, Inc. Qiagen, QIAvac and Vacman are all trademarks of their respective companies. PCR is a patented process of Hoffman-La Roche. Use of the PCR process requires a license.

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